The roles of PIN1 in the pituitary ganadotropes

THE ROLES OF PIN1 IN THE PITUITARY GONADOTROPES Luo Zhuojuan NATIONAL UNIVERSITY OF SINGAPORE 2010 THE ROLES OF PIN1 IN THE PITUITARY GONADOTROPES Luo Zhuojuan (Master of Science, Southeast University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENTS I would like to thank many people for their help and support during my doctoral research in National University of Singapore. This thesis could not be completed without them. My foremost and deepest gratitude to my supervisor A/P Philippa Melamed and my co-supervisor Dr. Yih-Cherng Liou for giving me the invaluable guidance and continuous encouragement throughout the study, for giving me generously their time and patience during my time under their supervision. Their support and understanding are deeply appreciated. My sincerest gratitude to my thesis committee, A/P Ge Ruowen and A/P Ng Huck Hui for giving me their comments and advices on this project. I have benefited a lot from their constructive insights. I would also like to extend my best gratitude to all other teaching staff and technical staff in Department of Biological Sciences for the good academic environment created. To my wonderful labmates, especially Andrea, Yingzi, Xia Yun, Qiaoyun, Ye Fan, Xiao Lin, Siew Hoon, Jiajun, Helen, Si Hui, Stefan, Cheng Yu, Jian Yuan, Lora and Jaw-Shin for giving me their great assistance, warm acceptance, as well as the friendship that will be cherished forever. To my husband for giving me endless love and his words of encouragement whenever I am down, for countless times giving me intellectual support that is truly valued. This path without him to walk with would never have been enjoyable. To my parents for always being there for me, for their unconditional support and love since the day I was created. To my parents-in-law for giving me their love and care, for all the moral support they provided. Last but not the least, I would like to thank National University of Singapore for providing me the Research Scholarship. And the work was financially supported by Ministry of Education Academic Research Fund (Singapore) Grant #R-154-000-410-112. i TABLE OF CONTENTS ACKNOWLEDGEMENTS .i TABLE OF CONTENTS ii SUMMARY v LIST OF TABLES vi LIST OF FIGURES .vii LIST OF FIGURES .vii LIST OF ABBREVIATIONS ix CHAPTER INTRODUCTION .1 1.1 Gonadotropins 1.1.1 Pituitary gland and gonadotropes 1.1.2 Gonadotropins and their biological functions .1 1.1.3 Regulation of gonadotropin synthesis and secretion 1.1.4 Immortalized murine gonadotrope cell lines 1.2 Basal and GnRH-induced gonadotropin subunit gene transcription 1.2.1 Transcriptional regulation of the αGSU subunit .7 1.2.2 Transcriptional regulation of the gonadotropin β subunits .10 1.2.3 Phosphorylation of transcription factors by MAPKs 15 1.3 Pin1 17 1.3.1 The identification of Pin1 .19 1.3.2 The uniqueness of Pin1 amongst peptidyl prolyl isomerases .19 1.3.3 The involvement of Pin1 in diverse biological and pathological processes .21 1.3.3.1 Pin1 and oncogenesis .23 1.3.3.2 Pin1 and degenerative disease .25 1.3.3.3 Pin1 and reproduction 26 1.3.4 The regulation of Pin1 activity .26 1.4 SF-1 27 1.4.1 Structure of SF-1 .29 1.4.2 Regulation of SF-1 transcriptional activity .31 1.4.2.1 Binding with cofactors .31 1.4.2.2 Post-translational modification of SF-1 .33 1.5 ATF3 35 1.5.1 Induction of ATF3 by multiple signals .35 1.5.2 Binding partners of ATF3 .37 1.5.3 Alternative splicing isoforms of ATF3 .39 1.5.4 Dichotomous role of ATF3 in oncogenesis 39 1.6 Ubiquitination and SUMOylation pathways 43 1.6.1 Protein regulation by ubiquitination .45 1.6.2 Protein regulation by SUMOylation .47 1.6.3 Crosstalk between ubiquitination and SUMOylation .49 ii 1.7 Hypothesis and aims 51 1.7.1 Hypothesis .51 1.7.2 Aims 51 CHAPTER MATERIALS AND METHODS 53 2.1 Cell culture .53 2.1.1 Growth conditions .53 2.1.2 Storing and recovery of cells 53 2.1.3 Treatment of cells .54 2.1.4 Transient transfection of cells .54 2.2 Plasmid constructs .57 2.2.1 Gonadotropin subunit gene promoter construct for luciferase assays 57 2.2.2 Expression vectors 57 2.2.3 Constructs for the two-hybrid assays 61 2.2.4 Verification by DNA sequencing 61 2.3 Antibodies 63 2.4 RNA-mediated knock-down of Pin1 expression .64 2.5 Reverse transcriptase (RT)-PCR analysis 65 2.5.1 RNA isolation .65 2.5.2 First strand cDNA synthesis .65 2.5.3 PCR and gel electrophoresis .66 2.5.4 Real-time PCR quantification analysis .68 2.6 Luciferase assay .70 2.6.1 Promoter study 70 2.6.2 Mammalian two-hybrid assay .71 2.7 Statistical analysis 71 2.8 Chromatin immunoprecipitation (ChIP) 72 2.9 Immunoprecipitation (IP) .75 2.10 In vivo ubiquitination assay .75 2.11 In vivo SUMOylation assay .76 2.12 Western blot .76 2.13 Fluorescence imaging 78 CHAPTER RESULTS .80 3.1 Pin1 induces gonadotropin β subunit gene transcription .80 3.1.1 Pin1 over-expression increases gonadotropin β subunit gene transcription .80 3.1.2 Pin1 knock-down decreases gonadotropin β subunit gene transcription 85 3.2 Pin1 is both transcriptionally and post-translationally regulated by GnRH 87 3.2.1 GnRH increases Pin1 protein levels .87 3.2.2 GnRH alters phosphorylation status of Pin1 .89 3.3 Pin1 is present on the promoters of the LHβ and FSHβ genes and interacts with various gene-specific transcription factors 93 3.3.1 Pin1 is present on the promoters of the LHβ and FSHβ genes .93 3.3.2 Pin1 interacts with various gene-specific transcription factors 95 iii 3.4 Pin1 increases transcriptional activity of SF-1, Pitx1 and Egr-1 .99 3.5 Effects of Pin1 on the levels of these transcription factors 102 3.6 Regulation of SF-1 ubiquitination .104 3.6.1 Ser 203 is required for SF-1 ubiquitination 104 3.6.2 Pin1 is required for SF-1 ubiquitination .106 3.7 GnRH treatment stimulates SF-1 ubiquitination 108 3.8 SF-1 can be ubiquitinated or SUMOylated at Lys 119 115 3.9 Poly-ubiquitin chains on SF-1 are assembled through either Lys 48 or Lys 63 of ubiquitin .117 3.10 SF-1 ubiquitination, but not SUMOylation, facilitates its interaction with Pitx1 119 3.11 Ubiquitination of SF-1 leads to its nuclear export .122 3.12 Pin1 targets SF-1 to increase its interaction with Pitx1 .124 3.13 Pin1 interacts with ATF3 .129 3.14 Pin1 increases the stability of ATF3 131 3.15 GnRH stimulates ATF3 transcription 133 3.16 Pin1, c-Jun and ATF3 form a complex in gonadotropes .135 3.17 ATF3 is SUMOylated 137 CHAPTER DISCUSSION AND CONCLUSIONS 140 4.1 Reproductive abnormalities in Pin1 knockout mice 140 4.2 Pin1 is involved in gonadotropin synthesis through its action on specific transcription factors .143 4.2.1 Pin1 is present on the promoters of both gonadotropin β subunit genes 145 4.2.2 Effect of Pin1 on the transactivation function of these transcription factors 146 4.2.3 Pin1 promotes ubiquitination of SF-1, which is required for SF-1 cofactor recruitment .147 4.2.4 Effect of Pin1 on protein stability of Pitx1 .152 4.3 Regulation of Pin1 by GnRH .153 4.3.1 Expression of Pin1 is induced by GnRH 154 4.3.2 Phosphorylation of Pin1 is regulated by GnRH 154 4.4 Regulation of SF-1 transcriptional activity by the crosstalk between various post-translational modifications .155 4.5 Interaction between Pin1 and ATF3 157 4.6 The Effect of Pin1 on ATF3 protein level .158 4.7 SUMOylation of ATF3 159 4.8 Conclusions 160 REFERENCES .162 iv SUMMARY Pin1 is a peptidyl-prolyl cis/trans isomerase which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds. Pin1 knockout mice have marked abnormalities in their reproductive development and function. However, the molecular mechanisms underlying their reproductive defects are poorly understood. In this study, it has been demonstrated that Pin1 is required for both basal and GnRH-induced gonadotropin β subunit gene transcription through physical and functional interactions with the transcription factors SF-1, Pitx1, and Egr-1. Pin1 activates transcription of the gonadotropin β subunit genes synergistically with these transcription factors, either by modulating their stability or by increasing their protein–protein interactions. Notably, it has been shown that Pin1 is required for the Ser 203 phosphorylation-dependent ubiquitination of SF-1, which facilitates SF-1–Pitx1 interactions and therefore results in an enhancement of SF-1 transcriptional activity. It has also been demonstrated that in gonadotrope cells sufficient levels of activated Pin1 are maintained through transcriptional and post-translational regulation by GnRH-induced signaling cascades. These results suggest that Pin1 functions as a novel player in GnRH-induced signal pathways and is involved in gonadotropin β subunit gene transcription by modulating the activity of various specific transcription factors. In addition, in this study it has been shown that Pin1 can complex with the αGSU gene transcription factors, c-Jun and ATF3, and increase the protein level of ATF3 in gonadotropes. These findings would lay the foundation for investigating whether Pin1 plays a role in transcriptional regulation of αGSU gene. v LIST OF TABLES Table 1.1: Overview of selected SF-1 target genes .28 Table 2.1: Optimized Lipofectamine 2000 (μL) : DNA (μg) 55 Table 2.2: Optimized GenePORTER (μL) : DNA (μg) 55 Table 2.3: Primer sequences for pCS2+ Pin1 and pCS2+ ATF3 constructs .58 Table 2.4: Primer sequences for tagged expression vectors 59 Table 2.5: Primer sequences for site-directed mutations .60 Table 2.6: Primers used for DNA sequencing .62 Table 2.7: Sequencing PCR reaction mixture 62 Table 2.8: Sequencing PCR parameters .63 Table 2.9: First strand cDNA synthesis reaction mixure .66 Table 2.10: Primers used for RT-PCR analysis .67 Table 2.11: PCR reaction mixure .67 Table 2.12: PCR parameters 68 Table 2.13: Primers used for real-time PCR analysis 69 Table 2.14: Real-time PCR reaction mixure 69 Table 2.15: Real-time PCR parameters .70 Table 2.16: Buffers used in ChIP analysis .74 Table 2.17: Primers used for amplification of the mouse LHβ and FSHβ promoters in ChIP .74 Table 2.18: Buffers used in Western blot 78 vi LIST OF FIGURES Fig 1.1: Schematic overview of the reproductive axis in male and female mammals .3 Fig 1.2: Schematic diagram of GnRH-induced signaling cascades .6 Fig 1.3: Schematic model of several elements defining the αGSU expression. Fig 1.4: Schematic models of transcription factors activating rodent gonadotropin β subunit gene expression. 10 Fig 1.5: Schematic model of the action of Pin1 in catalyzing cis-trans isomerization of pSer/Thr-Pro motif .18 Fig 1.6: The involvement of Pin1 in various cellualr processes and functional targets of Pin1 22 Fig 1.7: Schematic model of key structural domains in SF-1 29 Fig 1.8: Overview of SF-1 interaction partners. 32 Fig 1.9: ATF3 is able to function as both a tumour suppressor and oncogenic protein. 40 Fig 1.10: Overview of ubiquitination and SUMOylation conjugation pathway 44 Fig 3.1: Pin1 over-expression increases promoter activity of gonadotropin β subunit genes. .81 Fig 3.2: Pin1 over-expression increases endogenous mRNA levels of gonadotropin β subunit genes 82 Fig 3.3: Wild type Pin1, but not its WW domain and PPIase domain mutants, increases endogenous mRNA levels of gonadotropin β subunit genes. 84 Fig 3.4: Pin1 knock-down decreases endogenous mRNA levels of gonadotropin β subunit genes 86 Fig 3.5: GnRH increases Pin1 protein levels. 88 Fig 3.6: GnRH alters the phosphorylation status of Pin1. .90 Fig 3.7: p-Pin1 is associated with calcineurin catalytic subunit A. .92 Fig 3.8: Pin1 is present on the proximal promoters of the LHβ and FSHβ genes. 94 Fig 3.9: Co-immunoprecipitation of Pin1 and various gonadotropin β subunit gene-specific transcription factors. 96 Fig 3.10: Interaction between Pin1 and various gonadotropin β subunit gene-specific transcription factors shown by mammalian two-hybrid assays. 96 Fig 3.11: Mutagenesis of Ser/Thr-Pro motifs in SF-1 and Pitx1 reduces the interaction between Pin1 and SF-1 or Pitx1 .98 Fig 3.12: Pin1 and SF-1 have a synergistic effect on the LHβ but not on the FSHβ gene promoter. .100 Fig 3.13: Pin1 and Pitx1 have a synergistic effect on the FSHβ but not on the LHβ gene promoter. .100 Fig 3.14: Pin1 and Egr-1 have a synergistic effect on the LHβ gene promoter .101 Fig 3.15: Effects of Pin1 on the levels of these transcription factors. .103 Fig 3.16: Ubiquitination of SF-1 requires Ser 203. .105 Fig 3.17: Ubiquitination of SF-1 requires Pin1. 107 vii Fig 3.18: GnRH treatment increases ubiquitination of SF-1. 108 Fig 3.19: U0126 treatment inhibits GnRH-induced SF-1 ubiquitination. .110 Fig 3.20: Roscovitine (ROS) treatment inhibits SF-1 ubiquitination. .112 Fig 3.21: Dominant negative mutant CDK7 inhibits SF-1 ubiquitination .114 Fig 3.22: SF-1 is ubiquitinated at Lys 119 .115 Fig 3.23: SF-1 is SUMOylated at Lys 119 and Lys 194 116 Fig 3.24: SF-1 is ubiquitinated via Lys 48- and Lys 63-linked poly-ubiquitin chains. 118 Fig 3.25: SF-1 ubiquitination facilitates its interaction with Pitx1 121 Fig 3.27: Interaction between SF-1 and Pitx1 decreases in the absence of Pin1 .125 Fig 3.28: Exogenous Pin1 rescues the interaction between SF-1 and Pitx1 in MEF Pin1 –/– cells 127 Fig 3.29: Mutation of Pin1 binding site in SF-1 reduces its interaction with Pitx1. .128 Fig 3.30: Interaction between Pin1 and ATF3 .130 Fig 3.31: Pin1 stabilizes ATF3. .132 Fig 3.32: GnRH upregulates ATF3 transcriptionally. .134 Fig 3.33: Pin1, c-Jun and ATF3 form a complex in gonadotropes 136 Fig 3.34: ATF3 is SUMOylated in LβT2 cells. .139 Fig 4.1: Model of regulation of gonadotropin β subunit gene transcription by Pin1.161  viii References characterization of phospholipids as ligands for the orphan nuclear receptor steroidogenic factor-1. 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Schematic model of the action of Pin1 in catalyzing cis-trans isomerization of pSer/Thr-Pro motif Reversible phosphorylation of proteins on Ser/Thr-Pro motifs by Pro-directed kinases produces the substrate for Pin1, which alters the conformation of target proteins by catalyzing the trans to cis or the cis to trans isomerization, depending on specific target sites The resulting functional changes of the target... expressed in the gonadotropes and thyrotropes of the pituitary and in the trophoblasts of the placenta Cell or tissue-specific expression of αGSU is determined by distinct sets of cis-acting elements along with their cognate binding factors (Maurer et al., 1999) Cis-elements residing in the promoter that contribute to gonadotrope-specific αGSU gene transcription include the E boxes, Pitx1 binding element,... Transcriptional regulation of the gonadotropin β subunits As synthesis of the β subunit is the rate limiting step in hormone synthesis, regulation of the gonadotropin β subunit genes is crucial in the production of the physiologically active hormone Similar to the αGSU gene, gonadotropin β subunit genes, LHβ and FSHβ, are also regulated by interplay between extrinsic signals and intrinsic regulatory elements... activate the FSHβ promoter include Lhx3 (West et al., 2004) and the Smad family (Suszko et al., 2003) Activin, a member of the transforming growth factor β (TGFβ) superfamily of ligands, induces phosphorylation of several Smad proteins allowing their translocation into the 14 Introduction nucleus and binding to Smad binding elements (SBE) on the FSHβ promoter, where the Smads help recruit other activators... Phosphorylation of transcription factors by MAPKs It has been well accepted that serine/threonine residues preceding a proline (Ser/Thr-Pro) form one of the major regulatory phosphorylation motifs Protein or peptide containing proline is able to form both the cis and trans isomers because the unique five-membered ring structure of the proline side chain has the ability to adopt either the cis or trans state of the. .. Sentrin specific protease Ser/Thr-Pro Serine/threonine residues preceding a proline SF-1 Steroidogenic factor-1 SIL SCL interrupting locus SIM SUMO interaction motif siRNA Small interfering RNA xiii Smad Sma and Mad related protein SMRT Silencing mediator of retinoic acid and thyroid hormone receptor SNIP1 Smad nuclear interacting protein 1 SNURF/RNF4 Small nuclear ring finger protein/ring finger protein... and Arg 69 in the PPIase domain sequester the proline and the peptide bond undergoing cis/trans isomerization in the phosphorylated Ser/Thr-Pro motif, and are involved in the catalysis (Ranganathan et al., 1997) Thus, the N-terminal WW domain and C-terminal PPIase domain together form a double-check mechanism conferring the unique substrate specificity of Pin1, reducing the energy barrier between cis... gonadotropin subunit gene expression GnRH signaling also induces the release of calcium from intracellular stores which activates calmodulin, stimulating downstream calmodulin kinases and the phosphatase calcineuin, which play a crucial role in the derepression of the FSHβ gene (Lim et al., 2007) 5 in the text (Adapted from Naor, 2009 with modification) activated through phosphorylation by these kinases These... in all pituitary cell types and is essential for both anterior pituitary development and the synthesis of most of the pituitary hormones including the gonadotropins and TSH (Tremblay et al., 1998; Melamed et al., 2002) Both the number of gonadotropes and thyrotropes, as well as the levels of αGSU, LHβ, FSHβ and TSHβ transcript and protein within the individual cells, are substantially reduced in Pitx1-null... angle Most of the isomerization occurs in a surface-accessible bend, coil or turn of the target protein, inducing protein conformational change, therefore modulating the stability and/or activity of the protein Conformational change mediated by cis/trans isomerization of proline after phosphorylation of Ser/Thr-Pro motif has been identified as an intrinsic regulatory switch to modulate the stability . decreases in the absence of Pin1 125 Fig 3.28: Exogenous Pin1 rescues the interaction between SF-1 and Pitx1 in MEF Pin1 –/– cells 127 Fig 3.29: Mutation of Pin1 binding site in SF-1 reduces its interaction. THE ROLES OF PIN1 IN THE PITUITARY GONADOTROPES Luo Zhuojuan NATIONAL UNIVERSITY OF SINGAPORE 2010 THE ROLES OF PIN1 IN THE PITUITARY GONADOTROPES. and ATF3, and increase the protein level of ATF3 in gonadotropes. These findings would lay the foundation for investigating whether Pin1 plays a role in transcriptional regulation of αGSU gene.